CN103509105B - The production technique of high active hirudin is gone out based on anion-exchange column high efficiency separation - Google Patents
The production technique of high active hirudin is gone out based on anion-exchange column high efficiency separation Download PDFInfo
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Abstract
The present invention is based on a kind of novel anionic exchange column (DEAE-silica gel separator column), realizes high efficiency separation purifying r-hirudin.The leech powder made with Guangxi Hirudinaria manillensis is starting material, belongs to field of traditional Chinese medicine pharmacy, relates to the extraction of animal effective constituent, and object solves the problem that r-hirudin extraction is complicated, cost is high, be difficult to suitability for industrialized production.The invention is characterized in the separation and purification system around this cheap DEAE-silica gel separator column newly, devise the novel process of r-hirudin separation and purification.This technique is simple and quick, and cost is low; And the r-hirudin lyophilized powder activity obtained is high, and the rate of recovery is high, and purity is high, long preservative period, can be the raw material that the industries such as food, protective foods, medicine or makeup provide safety, high-quality.
Description
Technical field
This technique belongs to the separation and purification of biological medicine, and the fields such as medicine is refining made by Chinese medicine, are specifically related to a kind of production technique going out high active hirudin based on novel anionic exchange column high efficiency separation.
Background technology
R-hirudin (hirudin) is a kind of protein be present in the sialisterium of hirudinaria manillensis (leech), there is the antithrombin activity of high special, Trombin inhibiting bound substrates, be up to now find the natural specific inhibitor of the strongest zymoplasm.1984, Haycraft found that Hirudo extract has blood coagulation resisting function.Jocoby in 1904 is separated first and obtains this active ingredient from Hementaria officianalis, and called after r-hirudin.Nineteen fifty-five, Markwardt, successfully from Hementaria officianalis head part from being purified into sterling r-hirudin, starting the biological chemistry and the pharmaceutical research that carry out basis, and determining that in 1970 r-hirudin is the specific inhibitor of zymoplasm.1986, Harvey etc. obtained the cDNA of r-hirudin, and hirudin gene is expressed successfully in multiple host subsequently.The polypeptide that natural hirudin is made up of 65 amino acid, its effect has the following aspects: 1. have blood coagulation resisting function.The r-hirudin of different sources and structure, its anticlotting mechanism is not quite similar.2. antithrombin effect.The antithrombin effect of r-hirudin sees European Hementaria officianalis element (hiNmedic5nalix) L1j, Asia r-hirudin (Hirudinaria manillensis) and India leech etc.R-hirudin carries out in two steps to the effect of zymoplasm, the electronegative polarity C-end portion of the on-catalytic Post section first making zymoplasm positively charged and r-hirudin produces electrostatic interaction and forms initial combination, this combination is reset rapidly by controlling diffusion subsequently, forms the mixture combined closely in same thrombin activity site.In this mixture, the C-end of water frog element is combined in the Fibrinogen recognition site of zymoplasm, and Trombin inhibiting is to fibrinogenic activation, and N-end then combines with catalyzed by thrombin site, the katalysis of Trombin inhibiting.3. antiplatelet effects.1992, Como etc. extracted one and are called Hirudinaria manillensis antiplatelet protein from Mexico leech, had specificity and suppressed collagen to arrive again the effect of platelet aggregation, and to other inductors (as blood, zymoplasm etc.) induced platelet aggregation unrestraint effect.4. fibrin degradation (former) effect.1984, Malin-conlco etc. extracted a kind of material being called hementin from the huge leech of Amazon.This material can the particular rib chain of fibrin degradation (former), has antithrombin activity and the platelet aggregation that can significantly suppress ADP induce, has obvious effect for what prevent thrombotic diseases.
R-hirudin or a kind of highly stable protein, extremely stable in the dry state, the change (l.47-12.9) that simple temperature raises (100 DEG C of water-baths) or pH value does not affect its vigor, deposit at some denaturing agent also highly stable in case, only have its vigor when temperature and pH value raise simultaneously just to start to decline.Under general room temperature, r-hirudin can stable existence 6 months in water, heats 15min and be not destroyed at 80 DEG C.Improve the pH value of solution, its stability then reduces.At 20 DEG C, in 0.lmol/LHCI or 0.1mol/LNaOH, Absorbable organic halogens 15min, through 80 DEG C of process 15min and complete deactivation under the condition of pH=13.
some technical schemes retrieved the production method of natural hirudin in prior art are as follows:
1. Chinese patent: 03113566.8 denomination of invention: the cultural method of Hirudinaria manillensis and the extractive technique of r-hirudin.Summary: the invention provides a kind of cultural method of Hirudinaria manillensis and the extracting method of r-hirudin, capture healthy and strong Hirudinaria manillensis by introduction fine provenance or field, carry out manual grading skill segmentation to tame and docile, feed, carry out artificial propagation, wait to raise to a certain extent, chemical substance is adopted to stimulate fresh and alive Hirudinaria manillensis to make it secrete saliva, then r-hirudin crude product is obtained to the further separating and filtering of its saliva, again r-hirudin crude product is carried out deep processing to obtain meeting medical r-hirudin, this Hirudinaria manillensis is higher than other kind Hirudinaria manillensis content containing anticoagulative substance, medicinal effect is good, extract r-hirudin method simple, after extracting r-hirudin, Hirudinaria manillensis is put back to pond cultivation, be separated by and can extract again again for one week or ten days, the fresh and alive Hirudinaria manillensis of every bar can extract 6-7 time repeatedly, each can extract r-hirudin crude product 6-7 milligram about, good product quality, economize on resources again, improve the utility value of Hirudinaria manillensis.
2. Chinese patent: 200610019531.0 denominations of invention: a kind of method and application taking hirudinaria manillensis as raw material and prepare r-hirudin.Summary: the invention discloses with hirudinaria manillensis is method and the application that r-hirudin prepared by raw material, the feature of this preparation method is to comprise hirudinaria manillensis slurrying, preparation suspension liquid and dry three steps, the powder obtained, it saves the topmost effective active composition of hirudinaria manillensis---natural hirudin.It is simple that the present invention has technique, and production cost is low, product long preservative period Matkwatdt zymoplasm direct titrimetric method can be adopted to detect natural hirudin content, thus makes steady quality, the features such as product curative effect is guaranteed.R-hirudin has wide range of applications, method by routine makes separately capsule, soft capsule, granule, tablet use, or make compound preparation with other animal and plant medicines, for preventing and treating the cardiovascular and cerebrovascular diseases such as cerebral apoplexy, hypertension, hyperlipidaemia, coronary heart disease.
3. Chinese patent: 200710026461.6 denominations of invention: a kind of is raw-material technique for producing hirudin with Hirudinaria manillensis saliva.Summary: a kind of is raw-material technique for producing hirudin with Hirudinaria manillensis saliva, belongs to the field of Chinese medicines, relates to the extraction of animal effective constituent.Object solves whose extraction process of courting death of tradition to be difficult to the problem of suitability for industrialized production.This technique extracts r-hirudin with Hirudinaria manillensis saliva, can suitability for industrialized production.Hirudinaria manillensis saliva normal saline extraction, extracting solution boils, filter, get filtrate.Solid substance normal saline extraction, filters, gets filtrate.Whole liquid before and after merging, add Lyphgel process twice, filter, get filtrate.Filtrate boils rear rapid cooling, refrigerated overnight.Centrifugal, after removing supernatant liquor concentrating under reduced pressure, lyophilize becomes lyophilized powder again.This production technique is simple, and product vigor is high, about having very strong thrombus dissolving.
4. Chinese patent: 200710026462.0 denominations of invention: a kind of is raw-material technique for producing hirudin with Hirudinaria manillensis frozen fresh body.Summary: a kind of is raw-material technique for producing hirudin with Hirudinaria manillensis frozen fresh body, belongs to the field of Chinese medicines, relates to the extraction of animal effective constituent.Object solves whose extraction process of courting death of tradition to be difficult to the problem of suitability for industrialized production.This technique adopts and extract r-hirudin from overall Hirudinaria manillensis, can suitability for industrialized production.First Hirudinaria manillensis homogenate, soup compound normal saline extraction three times, filtration.Filtrate is boiled, filter removal foreign protein.Collect filtrate, through freeze concentration, after alcohol precipitation, obtain r-hirudin crude product.Use brine r-hirudin crude product again, then concentrated cleaning solution, last lyophilize obtains product.Technique for producing hirudin method is simple, and final product quality is qualified.The pharmacological results shows, have good pharmacological action with the leech that this technique is extracted.
5. Chinese patent: 200710066096.1 denominations of invention: a kind of extracting method of effective constituent of natural hirudin.Summary: the extracting method that the invention discloses a kind of active ingredient of natural leech essence, after adding alcohol solution dipping 30-50 hour of content 15 ~ 25%, filters, obtains filtrate and the dregs of a decoction after using water cleaning hirudinaria manillensis clean; Rubbed by the Hirudinaria manillensis dregs of a decoction, then the aqueous acetone solution adding content 15 ~ 25% soaks, and filters, obtains filtrate, after respectively twice filter vacuum being reclaimed ethanol and acetone, merging filtrate, conventional vacuum lyophilize, obtains product.Present invention process is simple, and with low cost, yield is higher, good product quality, long preservative period, and whole production process is without " three wastes " discharge, and product is as the raw material in medicine, beauty treatment, the large field of protective foods three.
6. Chinese patent; 201110237495.6 denomination of invention: a kind of production method of natural hirudin.Summary: the production method that the invention discloses a kind of natural hirudin, that the feature of this production method is to comprise is heavy containing the extraction of natural hirudin liquid, acid, heating, dehydration and drying and other steps, the natural hirudin powder obtained, Warkawardt zymoplasm direct titrimetric method can be adopted to detect the content of its activeconstituents, make steady quality, product curative effect is guaranteed.The method has the features such as technique is simple, extraction yield is high, production cost is low, product purity is high, long preservative period, can be the raw material that food, protective foods, medicine or makeup provide safety, high-quality.
Recognize from the above-mentioned documents and materials retrieved, current studies in China extracts the method for r-hirudin, is mostly to be rubbed by leech or dry rear water, ethanol, ultrafiltration or chromatography extraction.But, aforesaid method has a lot of weak point, and after mainly extracting, effective constituent is not high, active general at about 150 ~ 6000ATU/g, and according to the active height of different extract not etc., foreign matter content is high, is not suitable for injecting drug use, and some extracting method are numerous and diverse, some procedures are slower, be not suitable for industrial production, so the extracting method of r-hirudin also has a lot of people in research at present, it is desirable to find the extracting method that efficiency is high, cost is low, activeconstituents is high.
Summary of the invention
This technique is to solve above-mentioned problems of the prior art, there is provided a kind of production technique going out high active hirudin based on novel anionic exchange column high efficiency separation, this process efficiency is high, cost is low, and it is high to obtain having purity, active high, the measured natural hirudin of matter.
For achieving the above object, the technical solution used in the present invention is as follows:
The production technique going out high active hirudin based on novel anionic exchange column high efficiency separation of the present invention, comprises the steps:
(1) from leech powder, slightly carry r-hirudin: add trichoroacetic acid(TCA) extracting solution according to 2 times of material weight, the centrifugal 30min of 8000r/min, supernatant is for subsequent use; Precipitation continues to repeat extraction twice, all supernatant liquor mixing, and regulate supernatant liquor about pH to 4.0, add three times of cold acetone precipitation, 4 DEG C of hold over night, reclaim the acetone in supernatant, precipitation goes acetone for subsequent use;
(2) DEAE-silicagel column separation and purification: the ultrapure water adding 1-10 times amount in above-mentioned steps (1) gained precipitation, fully dissolves, carries out separation and purification with DEAE-silicagel column medium pressure liquid chromatography, and collect r-hirudin peak position elutriant, for subsequent use;
(3) desalination and concentration: gained supernatant liquor in step (2) is dialysed, and water is changed 2-8 time in centre, and every minor tick is not less than 2h; The supernatant liquor of dialysing is concentrated by rotary evaporation (40-70 DEG C, 0-250r/min), and 0-8 DEG C of refrigeration is for subsequent use;
(4) in Freeze Drying Equipment freeze-drying, after collection, preservation is sealed.
The filler of the DEAE-silicagel column described in described step (2) is macroporous silica gel, and aperture is at 300-600, and its surface-coated one deck is containing the polymkeric substance of diethyl amido, and filler granularity is 30-150 μm.
The described polymkeric substance containing diethyl amido is the Mierocrystalline cellulose that methacrylic acid diethylamino second lipopolymer and diethylamino ethyl replace.
In described step (2), the moving phase of DEAE-silicagel column separation and purification is pure salts solution, nontoxic pollution-free, and flow rate of mobile phase is high, and separation efficiency is good, and column temperature is normal temperature, adopts UV-detector to carry out liquid chromatographic detection to sample solution.
Dialysis described in step (3) is the dialysis membrane used is the dialysis tubing that molecular weight cut-off is less than or equal to 7000, the minimum 24h of dialysis time; Concentrated is the moisture rotating evaporative removal 30% ~ 80% under 40-70 DEG C of scope, obtains concentrated solution.
Can be combined with Thrombin specificity according to r-hirudin, make thrombin inactivation, it is the principle of 1:1 in conjunction with ratio, can detect the activity of natural hirudin by Matkwardt zymoplasm direct titrimetric method.
The measure unit of r-hirudin is international unit, represents with ATU, and the international unit of thrombin activity is NIH, during namely 1 ATU equals and the r-hirudin amount of 1 NIH zymoplasm.
That component that in natural leech powder, anticoagulant active is the highest is considered to r-hirudin usually.The r-hirudin powder product that this technique obtains, can adopt Markwardt zymoplasm direct titrimetric method to detect its anticoagulant active.
Natural hirudin
activedetection method:
Accurately take this product powder 0.01g, be placed in 1.5ml centrifuge tube, add 400 μ L 0.9% normal saline solutions, shake up and make it to dissolve, be mixed with 0.025g/ml need testing solution.Precision measures need testing solution 100 μ L, put in 1.5ml centrifuge tube, add three strongest ones' aminomethane hydrochloride buffer (PH7.4) the 200 μ L containing 0.5% human fibrinogen, shake up, put in water-bath (37 ± 0.5) DEG C, slowly drip in every 1ml() containing 40NIH(unit) thrombin solution (5 μ L/min, dropping limit, limit shakes up gently) to occurring in solution solidifying, record consumes the volume of thrombin solution.Be calculated as follows
active:
U = C1V1 / (C2V2 )
In formula: U is the activity unit (ATU/g) of every 1 g r-hirudin;
C1 is the concentration (NIH/mL) of thrombin solution;
C2 is the mass concentration (g/mL) of need testing solution;
V1 is the volume (μ L) consuming thrombin solution;
V2 is the add-on (μ L) of need testing solution;
Above-mentioned natural hirudin activity test method is all applicable to the quality examination of the various products of natural hirudin made by main raw material.
Technique of the present invention is simple, extraction yield is high, production cost is low, product purity is high, long preservative period, Markwardt zymoplasm direct titrimetric method can be adopted to detect its anticoagulant active, particularly the present invention adopt extraction unlike the prior art, precipitation, separation and purification and drying technique, thus make quality more stable, easy to carry and use, can be the raw material that food, protective foods, medicine or makeup provide safety, high-quality, in clinical application, according to different needs, the present invention can be made pointed product.
Natural hirudin purity through inventive method purifying gained of the present invention is high, and active high, cost is low, simple to operate, easily repeats, and can carry out suitability for industrialized production.
The natural hirudin product that this technique obtains may be used for the raw material of food, healthcare products, medicine or makeup, effects such as there is anticoagulant and thrombolytic, improve blood circulation, enhance metabolism, clinically for preventing and treating cardiovascular and cerebrovascular diseases, as apoplexy, coronary heart disease, hyperlipidemia etc.
Accompanying drawing explanation
Fig. 1 is the HPLC figure of leech powder crude extract, can find out, in leech powder except r-hirudin, also containing a large amount of foreign proteins, causes the difficulty extracting high purity r-hirudin larger.Through repeatedly duplicate detection, and scheme (Fig. 3) comparative analysis with the r-hirudin purified product HPLC that Nanning Jin Haikekang Pharmaceutical Technology Co., Ltd provides and learn No. 9 protein peaks and r-hirudin purified product peak.
Fig. 2 is the color atlas with DEAE-column chromatography on silica gel column separating purification r-hirudin extracting solution, still contain a large amount of foreign proteins in crude extract as seen from the figure, but No. 3 peaks and other albumen can be separated well with selected DEAE-column chromatography on silica gel post.Collect four peak position protein solutions, dialysis also adopts Markwardt zymoplasm direct titrimetric method detection of active after freeze-drying, find that No. 3 peak protein anticoagulant activity are maximum, average at about 10000ATU/g, the hirudin activity of all the other absorption peak position albumen is all less than 400ATU/g, so No. 3 protein peaks and r-hirudin purified product peak, the r-hirudin purified product of collection liquid namely this experiment of this part.
The r-hirudin purified product HPLC that Fig. 3 provides for Nanning Jin Haikekang Pharmaceutical Technology Co., Ltd schemes, and in figure, No. 6 peaks are r-hirudin purified product peak.
Fig. 4 is the HPLC figure of the r-hirudin purified product obtained by this technique, and in figure, No. 2 peaks are r-hirudin purified product peak, can find out that the r-hirudin purity through purifying is very high.Contrast can find out with Fig. 3, taller through this technique sterling purity that the r-hirudin purified product that obtains provides than the said firm of purifying.
HPLC condition:
Chromatographic column: TSK2000 7.8 × 300mm detector: UV 210nm
Moving phase: 0.1M KH
2pO
40.1M (NH
4)
2sO
4pH=6.7 column temperature: 20 DEG C
Sample size: 10 μ L flow velocity: 1mL/min.
DEAE-column chromatography on silica gel lives condition:
Chromatographic column: DEAE-column chromatography on silica gel post 20 × 100mm detector: UV 210nm
Moving phase: A liquid: 10mM Tris PH=7.0 B liquid: 10mM Tris 1.0M NaCl PH=7.0
Sample size: 0.5mL flow velocity: 5mL/min.
Gradient: 0% B 5min 0-50%B 30min column temperature: 20 DEG C.
Embodiment
Embodiment one:
Take leech powder powder (active the is 300ATU/g) 20g that Jin Haikekang Pharmaceutical Technology Co., Ltd of Nanning City produces, add ultrapure water 20ml, 5%TCA (PH 1.15) 20ml, the centrifugal 20min of 8000r/min, supernatant is for subsequent use; Precipitation continues to repeat extraction twice, and all supernatant liquor mixing, regulate pH to 4.0, add three times of cold acetone precipitation, hold over night, HPLC detected result is shown in Fig. 1.
DEAE-silicagel column liquid-phase chromatographic column (20 × 100mm) separation and purification, chromatographic column stopping composition is the macroporous silica gel that surface-coated has one deck methacrylic acid diethylin second lipopolymer, and macroporous silica gel aperture 300, filler granularity is 30 μm.
UV-detector is adopted to carry out liquid chromatographic detection to sample solution, determined wavelength 210nm, flow velocity 5mL/min, gradient: 0%B (A:10mM Tris-HCl PH=7.0, B:10mM Tris-HCl 1.0M NaCl PH=7.0) 8min, 0-50%B 30min, 0%B 10min, collects r-hirudin peak position elutriant, dialysis 24h, water is changed 5 times in centre, and every minor tick is greater than 2h.Dialyzate rotary evaporation (47 DEG C) falls the moisture of 50%, freeze-drying, collects, and sealing is preserved, and detected result is shown in Fig. 2, Fig. 4.The activity that the r-hirudin purified product that present method obtains detects this product r-hirudin purified product through employing Markwardt zymoplasm direct titrimetric method is 11600ATU/g, and yield is 1.12%.
Embodiment two:
Take leech powder powder (active the is 160ATU/g) 20g that Jin Haikekang Pharmaceutical Technology Co., Ltd of Nanning City produces, add ultrapure water 20ml, 5%TCA (PH 1.15) 20ml, the centrifugal 15min of 8000r/min, supernatant is for subsequent use; Precipitation continues to repeat extraction twice, and all supernatant liquor mixing, regulate pH to 4.0, add three times of cold acetone precipitation, hold over night, HPLC detected result is shown in Fig. 1.
DEAE-silicagel column liquid-phase chromatographic column (20 × 100mm) separation and purification, chromatographic column stopping composition is the cellulosic macroporous silica gel that surface-coated has one deck diethylamino ethyl and replaces, and macroporous silica gel aperture 400, filler granularity is 60 μm.
UV-detector is adopted to carry out liquid chromatographic detection to sample solution, determined wavelength 210nm, flow velocity 5mL/min, gradient: 0%B (A:10mM Tris-HCl PH=7.0, B:10mM Tris-HCl 1.0M NaCl PH=7.0) 5min, 0-60%B 30min, 0%B 10min, collects r-hirudin purified product peak position elutriant, dialysis 24h, water is changed 5 times in centre, and every minor tick is greater than 2h.Dialyzate rotary evaporation (47 DEG C) falls the moisture of 60%, freeze-drying, collects, and sealing is preserved, and detected result is shown in Fig. 2, Fig. 4.The activity that the r-hirudin purified product that present method obtains detects this product r-hirudin through employing Markwardt zymoplasm direct titrimetric method is 6560ATU/g, and yield is 0.24%.
Embodiment three:
Take leech powder powder (active the is 160ATU/g) 20g that Jin Haikekang Pharmaceutical Technology Co., Ltd of Nanning City produces, add ultrapure water 20ml, 5%TCA (PH 1.15) 20ml, the centrifugal 15min of 8000r/min, supernatant is for subsequent use; Precipitation continues to repeat extraction twice, and all supernatant liquor mixing, regulate pH to 4.0, add three times of cold acetone precipitation, hold over night, HPLC detected result is shown in Fig. 1.
DEAE-silicagel column liquid-phase chromatographic column (20 × 100mm) separation and purification, chromatographic column stopping composition is the macroporous silica gel that surface-coated has one deck methacrylic acid diethylin second lipopolymer, and macroporous silica gel aperture 500, filler granularity is 100 μm.
UV-detector is adopted to carry out liquid chromatographic detection to sample solution, determined wavelength 210nm, flow velocity 5mL/min, gradient: 0%B (A:10mM Tris-HCl PH=7.0, B:10mM Tris-HCl 1.0M NaCl PH=7.0) 5min, 0-60%B 30min, 0%B 10min, collects r-hirudin purified product peak position elutriant, dialysis 24h, water is changed 5 times in centre, and every minor tick is greater than 2h.Dialyzate rotary evaporation (47 DEG C) falls the moisture of 60%, freeze-drying, collects, and sealing is preserved, and detected result is shown in Fig. 2, Fig. 4.The activity that the r-hirudin purified product that present method obtains detects this product r-hirudin purified product through employing Markwardt zymoplasm direct titrimetric method is 6580ATU/g, and yield is 0.23%.
Claims (3)
1. go out a production technique for high active hirudin based on anion-exchange column high efficiency separation, it is characterized in that comprising the steps:
(1) from leech powder, slightly r-hirudin is carried: doubly add trichoroacetic acid(TCA) extracting solution according to the 2-6 of material weight, centrifugal, supernatant is for subsequent use; Precipitation continues to repeat extraction twice, all supernatant liquor mixing, and regulate supernatant liquor about pH to 4.0, add three times of cold acetone precipitation, 0-8 DEG C of hold over night, reclaim the acetone in supernatant, precipitation goes acetone for subsequent use;
(2) DEAE-silicagel column separation and purification: add in above-mentioned steps (1) gained precipitation
1-10the ultrapure water of times amount, fully dissolves, and carries out separation and purification with DEAE-silicagel column medium pressure liquid chromatography, and collects r-hirudin peak position elutriant, for subsequent use;
(3) desalination and concentration: gained supernatant liquor in step (2) is dialysed, and water is changed 2-8 time in centre, and every minor tick is not less than 2h; The supernatant liquor of dialysing is concentrated by rotary evaporation (40-70 DEG C, 0-250r/min), and 0-8 DEG C of refrigeration is for subsequent use;
(4) in Freeze Drying Equipment freeze-drying, after collection, preservation is sealed;
The filler of the DEAE-silicagel column described in step (2) is macroporous silica gel, and aperture is 300-600, and its surface-coated one deck is containing the polymkeric substance of diethylamide, and filler granularity is 30-150 μm;
The described polymkeric substance containing diethylamide is the Mierocrystalline cellulose that methacrylic acid diethylamino second lipopolymer and diethylamino ethyl replace.
2. the production technique going out high active hirudin based on anion-exchange column high efficiency separation according to claim 1, it is characterized in that: be separated elution process easy, environmentally safe, in described step (2), the separation and purification of DEAE-silicagel column is by regulating the ionic concn of moving phase to carry out gradient elution, column temperature is normal temperature, adopts UV-detector to carry out liquid chromatographic detection to sample solution.
3. the production technique going out high active hirudin based on anion-exchange column high efficiency separation according to claim 1, it is characterized in that: dialysis described in step (3) is the dialysis membrane used is the dialysis tubing that molecular weight cut-off is less than or equal to 7000, the minimum 24h of dialysis time; Concentrated is the moisture rotating evaporative removal 30% ~ 80% under 40-70 DEG C of scope, obtains concentrated solution.
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| CN113080961B (en) * | 2021-03-16 | 2023-02-10 | 宁波博睿瀚达生物科技有限公司 | Endotoxin-free hirudin anticoagulation vacuum blood collection tube and preparation method thereof |
| CN113072638B (en) * | 2021-03-16 | 2022-12-23 | 宁波博睿瀚达生物科技有限公司 | Method for removing endotoxin in recombinant hirudin protein solution |
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